Detecting the presence of target nucleic acids is vital in numerous applications in medical diagnostics, forensics, genetic analysis, and public health. For instance, identifying specific DNA sequences is critical for diagnosing inherited disorders, determining susceptibility to disease, and identifying causal agents of infectious diseases. Polymerase chain reaction (PCR) provides a highly sensitive method for detecting the presence of target nucleic acids by selective amplification of target nucleic acids. The method relies on use of oligonucleotide primers that hybridize to opposite ends of a target nucleic acid segment, an amplicon, and prime copying of the nucleic acid segment by a polymerase. Reiterative rounds of DNA synthesis, denaturation, and reannealing allows exponential amplification of a given target nucleic.
Primer selection is a major determinant in the success or failure of the amplification reaction. Critical factors include primer length, melting temperature (Tm), sequence specificity, complementary primer sequences, G/C content, and 3′-terminal region sequence. In general, primers must hybridize with specificity to the target nucleic acid but not hybridize to and amplify non-target nucleic acid sequences.
Amplifying non-target nucleic acids becomes problematic when the 3′ end of a primer is complementary to another primer. These primers will tend to hybridize to each other, which are then extended by polymerase to form “primer-dimer” products. Subsequent amplification of primer-dimers leads to depletion of primers, resulting in reduced sensitivity or even failure to amplify the intended target nucleic acid. Performing primer extensions or preamplification annealing at temperatures limiting primer-primer hybrids (e.g., “hot start” PCR, D'Aquila, R. T. et al., Nucleic Acids Res. 19: 3749 (1991);“touchdown” PCR, Don, R. H. et al., Nucleic Acids Res. 19: 4008 (1991)) or adjusting buffer components to increase hybridization stringency may minimize primer-dimer interference. However, the presence of excess primers during PCR reactions allows even weak complementarity at the 3′ terminal region to generate these interfering side products.
Although choosing different regions of the target nucleic acid for selecting primers provides a basis for limiting non-target nucleic acid dependent amplifications, constraints on selecting sequences for generating primers can limit the choice of alternative primer designs. For example, directly amplifying short, tandem repetitive sequences such as telomere repeats is difficult since the primers for these sequences will always have some degree of complementarity. These repetitive sequences do not normally afford a choice in primer sequences for limiting formation of primer-dimer products. Consequently, current methods for estimating telomere lengths rely on restriction enzyme digestion of genomic DNA followed by hybridization with repeat sequences (terminal restriction fragment analysis; see Harley, C. B. et al, Nature 345: 458-460 (1990)), indirect amplification of repeats using unique sequences positioned outside of the repeat region (see Kozlowski, M. R. et al., U.S. Pat. No. 5,741,677), fluorescence in situ hybridization (see Henderson, S. J. Cell Biol. 134: 1-12 (1996)), or flow cytometry methods (see Hultdin, M. Nucleic Acids Res. 26: 3651-3656 (1998)). Generally, these procedures are time consuming or require substantial quantities of DNA. Since the copy number of telomere repeats and other tandemly repetitive sequences in a cell correlate with the physiological or diseased states of a cell, there is a need for compositions and methods for rapidly amplifying and quantitating these sequences while generally avoiding competing primer-dimer side reactions during amplification.